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Griffith Research Online
https://research-repository.griffith.edu.au
A Modified MS2 Bacteriophage PlaqueReduction Assay for the
RapidScreening of Antiviral Plant Extracts
AuthorCock, Ian, Kalt, Fred
Published2010
Journal TitlePharmacognosy Research
DOI https://doi.org/10.4103/0974-8490.69108
Copyright StatementCopyright 2010 Phcog.net. The attached file
is reproduced here in accordance with the copyright policyof the
publisher. Please refer to the journal's website for access to the
definitive, published version.
Downloaded fromhttp://hdl.handle.net/10072/34405
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Pharmacognosy Research | July 2010 | Vol 2 | Issue 4 221
Address for correspondence:Dr. Ian Cock, Biomolecular and
Biomedical Sciences, Griffith University, Nathan Campus, 170
Kessels Road, Nathan, Queensland 4111, Australia, E-mail:
[email protected]
DOI: 10.4103/0974-8490.69108
A modified MS2 bacteriophage plaque reduction assay for the
rapid screening of antiviral plant extractsIan Cock, F. R.
Kalt1
Biomolecular and Physical Sciences, Nathan Campus, Griffith
University, 170 Kessels Road, Nathan, Queensland 4111, 1Genomics
Research Center, Gold Coast Campus, Griffith University, Parklands
Drive, Southport, Queensland 4222, Australia
Submitted: 05-01-2010 Revised: 24-01-2010 Published:
07-09-2010
O R I G I N A L A R T I C L E
INTRODUCTION
The market for antiviral drugs is estimated at approximately US$
20 billion per year and this figure is expected to increase as new
antiviral agents become available. Following the recent worldwide
H1N1 influenza outbreak, sales of antiviral drugs (especially
oseltamivir phosphate (Tamiflu)) are expected to vastly increase
during the 2009 – 2010 period. In 1990, there were only five
licensed antiviral drugs; today this figure has grown to over
60.[1] However, most of these drugs are targeted at human
immunodeficiency virus (HIV) and various herpes viruses. There is
an urgent need to develop therapies against the myriad of viral
diseases for which no therapies currently exist. As most medically
important human viruses are RNA viruses,[2] the discovery of agents
directed against RNA viruses is particularly important.
Discovery and development of effective antiviral agents is a
difficult task and has had limited success. As viruses use host
cells to replicate, finding targets for drugs that eliminate the
virus without harming the host cells is vital. The plant kingdom
contains many unique unclassified compounds that are yet to be
screened for anti-viral properties and may provide drug candidates
for the treatment of viral diseases. For these agents to be
successful as anti-viral agents, limited toxicity toward human
cells is necessary.
The development of safe, cheap, rapid, high throughput assays is
essential for the discovery of antiviral drugs from plants.
Currently, most assays are based on virus-induced cytopathic effect
reduction assays (CPE-RA)[3-5] or genomic/subgenomic replicon-based
assays.[6-8] Although the CPE-RA and replicon-based assays are
specific for viruses and cell lines, they are expensive, time
consuming, and require specialized equipment and training. Both
these methods involve growing cell lines for several days,
inoculating them with the test virus, treating them with the
potential antiviral agent, and after several more days, checking
for a response (eg. reduction of the cytopathic effect or
inhibition of viral replication). The time required for these
assays and/or their low throughput nature limit
Introduction: Traditional methods of screening plant extracts
and purified components for antiviral activity require up to a week
to perform, prompting the need to develop more rapid quantitative
methods to measure the ability of plant based preparations to block
viral replication. We describe an adaption of an MS2 plaque
reduction assay for use in S. aureus. Results: MS2 bacteriophage
was capable of infecting and replicating in B. cereus, S. aureus
and F+ E. coli but not F- E. coli. Indeed, both B. cereus and S.
aureus were more sensitive to MS2 induced lysis than F+ E. coli.
When MS2 bacteriophage was mixed with Camellia sinensis extract (1
mg/ml), Scaevola spinescens extract (1 mg/ml) or Aloe barbadensis
juice and the mixtures inoculated into S. aureus, the formation of
plaques was reduced to 8.9 ± 3.8%, 5.4 ± 2.4% and 72.7 ± 20.9% of
the untreated MS2 control values respectively. Conclusions: The
ability of the MS2 plaque reduction assay to detect antiviral
activity in these known antiviral plant preparations indicates its
suitability as an antiviral screening tool. An advantage of this
assay compared with traditionally used cytopathic effect reduction
assays and replicon based assays is the more rapid acquisition of
results. Antiviral activity was detected within 24 h of the start
of testing. The MS2 assay is also inexpensive and non-pathogenic to
humans making it ideal for initial screening studies or as a
simulant for pathogenic viruses.
Key words: Aloe vera, antiviral assay, Camellia sinensis, MS2
bacteriophage, plaque reduction assay, Scaevola spinescens
A B S T R A C T
P H C O G R E S .
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222 Pharmacognosy Research | July 2010 | Vol 2 | Issue 4
their use for initial screening of large sample groupings.
Instead, CPE-RA and genomic/subgenomic replicon-based assays are
more suited for directed antiviral analysis.
Thus, there is a need to develop alternative preliminary
screening methods to be used prior to the cell screening assays.
These methods should be more rapid, cheap, have equal or greater
sensitivity, provide high levels of efficiency, and be safe for use
in the laboratory. For these reasons MS2 bacteriophage was chosen
for the development of a plaque reduction bioassay as an initial
anti-viral screening tool.
MS2 bacteriophage (family Leviviridae) is a small (27 – 34 nm)
icosahedral bacteriophage, which is usually described as an F
pilin/male-specific bacteriophage of Escherichia coli.[9] Indeed,
no evidence was found in the literature of MS2, infecting bacteria
other than F+ E. coli. The MS2 genome consists of a single sense
(+) RNA strand, 3569 nucleotides long, which contains four genes
[Figure 1]. These encode proteins necessary for phage maturation,
encapsidation, lysis of the bacterial host, and bacteriophage RNA
replication.[10,11]
MS2 bacteriophage is an attractive virus for the development of
an assay to screen for viral inactivation as most medically
important human viruses are RNA viruses.[2] MS2 phage resembles the
structure and function of many human enteroviruses (polioviruses
1-3, coxsakieviruses, echoviruses, and enteroviruses 68-72)[12]
making it a relevant model system. It is readily cultivatable in
titres up to 1012 pfu/ml and enumeration is rapid (less than 24
hours), and it is inexpensive. Furthermore, MS2 in not pathogenic
to humans and can therefore be tested in high numbers without the
need for additional safety measures. This makes MS2 bacteriophage a
useful simulant in place of small, human infective RNA viruses
(eg., Ebola virus, Marburg virus, and equine encephalitis
alphaviruses).
The current studies were undertaken to adapt an MS2
plaque reduction assay that is routinely used to test
environmental water quality[12] and to optimize the assay for
routine screening of plant extracts. To test this assay we chose to
examine plants that have previously been shown to inhibit viral
growth to determine whether antiviral activity could also be
detected in the plaque reduction assay. Recent studies have shown
that Camellia sinensis (tea) inhibits influenza virus replication
in the Madin-Darby canine kidney (MDCK) cell line,[13] blocks
attachment of HIV in T cells,[14] and has been reported to be
effective in the treatment of human papillomavirus (HPV)-induced
genital warts.[15] A previous study[16] found Scaevola spinescens
leaves to be capable of inhibiting greater than 25% of human
cytomegalovirus (CMV) production. Similarly, Aloe barbadensis
Miller extracts have also been shown to inhibit CMV production in
human cell lines.[17] Furthermore, aloe emodin, purified from A.
barbadensis, has been shown to inactivate herpes simplex virus type
1 and type 2, the varicella-zoster virus, pseudorabies virus, and
influenza virus.[18] In this report, we outline the use of the MS2
plaque reduction assay to detect antiviral activity in C. sinensis,
S. spinescens extracts, and A. barbadensis juice.
MATERIALS AND METHODS
Viral and Bacterial StocksMS2 bacteriophage, F+, and F- Amp+ E.
coli used in this study were supplied by Dr. Jatinder Sidhu and Dr.
Simon Toze of CSIRO, St. Lucia Qld, Australia. Bacillus cereus and
Staphylococcus aureus were obtained from Michelle Mendell and
Tarita Morais, Griffith University. All stock cultures were
subcultured and maintained in nutrient broth at 4oC.
Production of MS2 virusOne hundred milliliters of nutrient broth
(25 g/l) containing ampicillin (100 µg/ml) was inoculated with
either 1 ml F+ Amp+ E. coli culture or 1 ml of F- Amp+ E. coli
culture and incubated overnight at 37°C. Parallel studies examined
the ability of B. cereus and S. aureus to
Figure 1: Genetic map of the MS2 bacteriophage. Nucleotide
positions for the start and end of each gene are noted in the
figure. With regard to the overlapping genes, the coat and
replicase genes are read in the same frame, whereas, the lysis gene
reading frame is +1 with respect to these genes.
Cock and Kalt: Modified MS2 bacteriophage plaque reduction
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produce MS2 bacteriophage. One milliliter of B. cereus or S.
aureus were inoculated into 100 ml of nutrient broth (25 g/l) and
incubated overnight at 37°C. The following day, flasks containing
30 ml of nutrient broth (containing 100 µg/ml ampicillin for E.
coli cultures or without ampicillin for B. cereus and S. aureus
cultures) were inoculated with 1 ml of the relevant culture and
incubated for two hours at 37°C and 160 rpm. Once the bacterial
cells had reached log phase, 1 ml of stock MS2 virus (containing
approximately 108 plaque forming units) was added and incubated
overnight at 35°C. The solution was centrifuged at 4000 rpm for 10
minutes and the supernatant was collected and passed through a 22
μm Sarstedt filter. All stock and working solutions were stored at
4°C until further use.
Determination of MS2 viruscDNA SynthesiscDNA synthesis was
carried out using an iScript Select cDNA Synthesis Kit, (Bio-Rad
Laboratories, Inc., USA) as per the manual instructions. Briefly, 1
µl reverse transcriptase, 4 µl 5 x iScript Select reaction mix, 1
µl random primers (hexamers), and 13 µl RNA samples were added to
the individual PCR tubes. A Biorad C1000 thermocycler reaction
program employing the following steps was used: Five minutes at
25ºC for primer annealing, 30 minutes at 42ºC for cDNA synthesis,
and a final incubation step of five minutes at 85ºC to deactivate
the reverse transcriptase.
cDNA Polymerase Chain Reaction AmplificationPolymerase chain
reaction (PCR) using an Invitrogen PCR SuperMix was performed using
the synthesized cDNA as a template. Briefly, 10 µl Master mix, 1 µl
primer mix containing 0.5 µl of forward primer (MS2-109 CAT AGG TCA
AAC CTC CTA GGA ATG), 0.5 µl reverse primer (MS2-21 TCC TGC TCA ACT
TCC TGT CGA G), and 9 µl of each cDNA preparation were added to the
reaction tubes. PCR was performed using a Biorad C1000 thermocycler
comprising of a denaturing step (95ºC, 30 seconds) annealing step
(58ºC, 30 seconds), and extension step (72ºC, 30 seconds) for 32
cycles, and a final extension step of 72ºC for five minutes
followed by a cooling step of 4ºC for 15 minutes.
Agarose Gel ElectrophoresisThe PCR products were run on 3%
Agarose gel against a positive control (fresh MS2 virus) in order
to determine whether the MS2 bacteriophage was produced by each of
the bacterial species tested.
Plant Test SamplesA. barbadensis juice was obtained from Aloe
Wellness Pty Ltd., Australia, and was stored at 4oC until use. C.
sinensis leaf extract was obtained by immersing a single
tea bag (Lipton) in 50 ml deionized water for four hours at room
temperature, with constant mixing. S. spinescens plant material was
provided by Jeannie Cargo of Outback Books (an online supplier of
S. spinescens tea) as pre-dried and coarse milled whole plant
material. One gram of plant material was extracted in deionized
water for four hours at room temperature with constant mixing.
Following extraction, the liquid was filtered using Whatman No. 54
filter paper, followed by rotary evaporation in an Eppendorf
concentrator 5301. The resultant dry extract was weighed and
redissolved in 10 ml deionized water.
Soft Agar OverlayA soft agar overlay was prepared to a final
concentration of 0.7% w/v Agar, 1% w/v Glucose, 1% w/v CaCl2
solution, and 1% w/v MgSO4, and autoclaved at 120°C for 20 minutes.
The soft agar overlay was allowed to cool to 65°C, and then
nalidixic acid was added to a final concentration of 0.4% w/v. The
overlay was used immediately for the MS2 plaque inhibition assay
described later in the text.
MS2 Plaque Inhibition Assay Prior to plating, 490 μl of crude
plant extract was inoculated with 10 μl of MS2 virus (containing
approximately 1010 plaque forming units/ml) and incubated overnight
at 4ºC. The solution was added to 500 μl B. cereus, E. coli or S.
aureus as required and incubated at 37ºC for 20 minutes. The
bacteria/virus/extract mixture was then added to 3 ml soft agar
overlay and immediately poured over pre-made agar plates (2.8% w/v
Agar). The plates were allowed to set for 15 minutes at room
temperature, inverted, and incubated overnight at 37ºC. The
following morning, the plaques were counted and the percentage
inhibition recorded. Serial dilution was used to determine the
antiviral strength of the samples where necessary. Nutrient broth
and deionized water were used as negative controls, while C.
sinensis extract, S. spinescens extract, A. barbadensis juice, and
UV irradiation (microwave of 10 μl virus only for 4 × 30 seconds)
were used as positive controls.
Statistical AnalysisData are expressed as the mean ± SD of at
least three independent experiments. The Paired t-test was used to
calculate the statistical significance between the control and
treated groups, with a P value < 0.05 considered to be
statistically significant.
RESULTS
MS2 production and lysis of various bacteria Early in this
study, MS2 virus production was tested in F+ E. coli, F- E. coli,
B. cereus, and S. aureus. Although no lysis was seen in F- E. coli
(as seen by the lack of bacterial
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224 Pharmacognosy Research | July 2010 | Vol 2 | Issue 4
cell debris in the culture) and limited lysis was seen in the F+
E. coli, both B. cereus and S. aureus were highly prone to
bacteriophage-induced lysis (as seen by the degree of bacterial
cell debris). This is an interesting finding as previous reports
have described MS2 bacteriophage as being specific to F+ E. coli.
MS2 production studies using the other bacterial species were
initially included as negative controls. The lysis in the B. cereus
and S. aureus cultures indicates that MS2 may not be as specific as
previously reported. Moreover, increased lysis in the B. cereus and
S. aureus cultures indicates that these bacteria are more sensitive
to MS2 lysis than are F+ E. coli, and could therefore be used to
develop a more sensitive bioassay than the current assay used to
measure plaque reduction activity of water samples.
To provide evidence that the lysis seen in B. cereus and S.
aureus cultures was due to MS2 bacteriophage infection/production,
cDNA was produced against RNA from the cell-free culture media and
this cDNA was amplified using primer sequences specific to the 5’
non-coding region of the MS2 bacteriophage. As seen in Figure 2,
culturing MS2 in F- E. coli (lane B) resulted in the production of
no MS2 cDNA. In contrast, MS2 phage cDNA was clearly evident in the
samples synthesized against the MS2/ F+ E. coli culture.
Interestingly, cDNA synthesized against both the S. aureus (lane F)
and B. cereus (lane G) MS2 cultures also clearly showed the
presence of phage cDNA, indicating that these bacteria were indeed
capable of MS2 production. It was therefore evident that MS2
bacteriophage is not F+ E. coli specific, as had been previously
described, but could infect a wider array of bacterial species.
Even as the levels of MS2 cDNA production appeared similar in F+ E.
coli,
B. cereus, and S. aureus in this study, the latter two bacterial
species were more sensitive to lysis than was F+ E. coli.
To develop a more sensitive assay, the ability of the MS2
bacteriophage to produce plaques in F+ E. coli, B. cereus, and S.
aureus was compared. Plaque production in F- E. coli was also
determined as a negative control. Table 1 shows the number of
plaques seen when 500 µl of undiluted MS2 (containing 1010 plaque
forming units/ml) or 500 µl of a 1 in 50 dilution of MS2
(containing 2 × 108 plaque forming units/ml) were incubated with
500 µl of the bacterial stocks and plated. No plaque counts were
available for the B. cereus assays as very little bacterial growth
was seen in the presence of MS2 at the levels tested. It is likely
that while MS2 production is effective in B. cereus, lysis of the
bacteria is so complete that B. cereus is unsuitable for use as a
test bacterium. Concentration-response testing of MS2 in B. cereus
(unpublished results) showed an all or nothing response. As the
levels of MS2 plaque forming units were further reduced to 5 × 107
plaque forming units/ml, the ability to inhibit bacterial growth
was lost. However, at the same concentration, no plaques were
seen.
MS2 was tested at the concentrations indicated. The number of
plaques seen was recorded as the mean ± standard deviation of at
least triplicate determinations.
In contrast, high plaque numbers were seen in both F+ E. coli
and S. aureus when they were incubated with 5 × 109 plate forming
units of MS2. Indeed, in both cases the number of plaques was too
many to count and was recorded as > 200. When a 1 in 50 dilution
of the MS2 stock (108 plate forming units) was used, the plaque
numbers on F+ E. coli plates decreased dramatically to
approximately 24 plaques. S. aureus was more sensitive to
MS2-induced lysis. When tested with the addition of 108 plaque
forming units of MS2, approximately 180 plaques were seen on the S.
aureus plates. The higher sensitivity of S. aureus to MS2
bacteriophage allowed for the detection of lower levels of the
phage, and hence a more sensitive assay. The potential antiviral
agents were tested against S. aureus in all remaining plaque
reduction assays. No plaques were seen when MS2 was tested against
F- E. coli.
Figure 2: 3% Agarose gel of PCR products from cDNA synthesized
against MS2 viral RNA. (A and H) DNA bp ladder, (B) MS2
bacteriophage produced in F- E. coli, (C and D) MS2 bacteriophage
obtained from separate CSIRO stocks, (E) MS2 bacteriophage produced
in F+ E. coli, (F) MS2 bacteriophage produced in S. aureus, and (G)
MS2 bacteriophage produced in B. cereus. Tests were performed in
triplicate and representative results are shown here.
Table 1: MS2 plaque counts in the test bacterial speciesBacteria
MS2 Added
5 × 109 pfu 108 pfuF+ E. coli > 200 23.6 ± 5.6F- E. coli 0
0B. cereus NBG* NBG*S. aureus > 200 178.3 ± 30.7
*NBG = no bacterial growth observed.
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Screening of plant extractsA. barbadensis juice, C. sinensis
extract (1 mg/ml), and S. spinescens extract (1 mg/ml) were tested
for the ability to inhibit MS2 plaque formation in S. aureus
[Figure 3]. Microwave irradiation (positive control) of MS2
bacteriophage [Figure 3f] completely destroyed the plaque forming
potential of the phage. All plant extracts tested also reduced the
plaque formation in S. aureus. Both C. sinensis leaf extract
[Figure 3c] and S. spinescens extract [Figure 3d] were particularly
effective at inhibiting MS2 plaque formation. A. barbadensis juice,
while also able to partially inhibit the formation of MS2 plaques,
was substantially less effective than the other plant extracts.
Figure 4 shows the plaque formation in the presence of plant
extracts as a percentage of plaque formation in the negative
controls. No difference was seen between the negative controls
(nutrient broth or water added to the MS2 bacteriophage instead of
juice/extract). All plant extracts produced a statistically
significant decrease in MS2 plaque production with C. sinensis and
S. spinescens extracts almost completely blocking the plaque
formation at a 1 mg/ml concentration.
The plant extracts were further tested over a range of
concentrations to determine the minimum concentration capable of
inhibiting 100% of the plaque formation (PI100) and the minimum
concentration capable of inhibiting 50% of the plaque formation
(PI50) [Table 2]. PI100 values were not obtained for A. barbadensis
juice or S. spinescens, as none of the tested concentrations of
these extracts was found to inhibit 100% of the plaque formation,
even when tested undiluted. In contrast, C. sinensis leaf extract
was capable of
totally blocking MS2 plaque formation at a concentration of
approximately 19.6 mg/ml. PI50 values were obtained for both C.
sinensis leaf extract (4.9 ± 1.6 mg/ml) and S. spinescens extract
(7.9 ± 2.6 mg/ml). No PI50 was obtained for A. barbadensis juice,
as even when it was undiluted, the plaque counts did not decrease
to 50% of the control value.
All PI100 and PI50 values are expressed as mg/ml ± standard
deviation. NPI denotes that PI100/PI50 has not been achieved. All
values are the means of at least triplicate determinations.
DISCUSSION
Understanding the mechanism of viral replication is not the only
key step toward the identification of effective drugs against a
virus. Development of rapid screening assays is also essential for
antiviral drug discovery. The current study describes the
development of an MS2 bacteriophage plaque reduction assay in S.
aureus. The rapid nature of this test and its ease/low cost
compared to other antiviral assay techniques makes it a valuable
tool for rapidly screening potential antiviral agents, to target
samples for more specific screens.
Figure 3: MS2 plaque reduction assay in an S. aureus lawn
following incubation of the MS2 with (A) nutrient broth (negative
control), (B) deionized water (negative control), (C) C. sinensis
leaf aqueous extract 1 mg/ml), (D) S. spinescens water extract (1
mg/ml), (E) A. barbadensis juice, (F) nutrient agar, and microwave
irradiation (positive control). All assays were performed in
triplicate and representative assays are shown.
Figure 4: MS2 plaque formation presented as a percentage of
untreated phage plaque formation following incubation of the MS2
phage with (A) nutrient media (negative control), (B) deionized
water (negative control), (C) C. sinensis leaf water extract (1
mg/ml), (D) S. spinescens water extract (1 mg/ml), (E) A.
barbadensis juice, (F) nutrient agar, and microwave irradiation
(positive control). All results were reported as the mean of
triplicate assays ± standard deviation. * indicates statistically
significant results.
Table 2: Minimum concentrations capable of inhibiting 100%
(PI100) and 50% (PI50) of the plaque formation for anti-viral plant
samplesMS2 Phage Treatment PI100 PI50C. sinesis water extract 19.6
± 6.5 4.9 ± 1.6S. spinescens water extract NPI 7.9 ± 2.6A.
barbadensis juice NPI NPI
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These studies demonstrate that S. aureus is more sensitive to
MS2-induced lysis than F+ E. coli. This was a surprising result as
previous studies refer to MS2 as an F+ E. coli-specific
bacteriophage. No studies were found in literature that discussed
MS2 conjugation or lysis of other bacterial species. It has been
demonstrated that MS2 phage uptake into E. coli cells is mediated
through binding to the F pilin protein in the F pili.[19] Only F+
(but not F-) E. coli cells can take up MS2 bacteriophage, resulting
in lysis. Indeed, F- E. coli cells were used in our studies as a
negative control. Presumably any bacteria with F pilin expressed in
their cell walls would be able to take up MS2. F+ plasmids can be
easily transferred from E. coli cell to E. coli cell by
conjugation.[19] It is also likely that a similar exchange is
possible between different bacterial species. It is therefore
surprising that there are no other reports of MS2 plaque formation
in other bacterial species.
The formation of pili on the surface of Gram-negative bacteria
has been studied in detail. In contrast, the pilus assembly
pathways in Gram-positive bacteria have yet to be fully
characterized. Gram-positive bacteria use cell wall peptidoglycan
as a surface organelle for the covalent attachment of proteins.[20]
This strategy involves sorting signals of surface protein
precursors and sortase (a transpeptidase that cleaves sorting
signals and links the C-terminus of surface proteins via an amide
bond to the peptidoglycan cross-bridge).[20] To further validate
the potential of S. aureus as a suitable host for the MS2
bacteriophage, it needs to be shown that S. aureus contains these
mechanisms and possesses the ability to form pili. Related
bacterial species (Streptococcus pyogenes and Streptococcus
pneumoniae) have already demonstrated the ability to form
pili.[21-23] Vegetative forms of B. cereus have also been reported
to form pili.[24]
The sortase enzyme (Sortase A), a housekeeping enzyme,
responsible for catalyzing cell-wall anchoring of surface proteins
was discovered in S. aureus and is present in all Gram-positive
bacterial genomes except in Mycobacterium and Microplasma.[25] Many
pathogens harbor additional sortases, which are involved in iron
acquisition, sporulation, and pilus assembly.[26] Sortases of the
class C family form the largest group and are often present in
multiple copies in a genome. These sortases are encoded together
with their substrates, which constitute various types of pili in
many pathogens.
S. aureus has been shown to possess two classes of sortases,
Class A and Class D.[26,27] B. cereus has been shown to contain all
four sortase subgroups.[27] As these sortases are responsible for
the formation of pili on the surface of Gram-positive bacteria,
this may explain the efficiency that the MS2 virus has in relation
to B. cereus and S. aureus.
Further tests examining the interaction of other sortase
containing bacteria with the MS2 virus are necessary to provide an
insight into bacterial lysis, sortase subfamily, and MS2
selectivity.
Although this assay was developed to test plant extracts for
antiviral activity, a similar assay utilizing E. coli is routinely
used to test environmental water quality.[12] The current study has
demonstrated greater MS2 sensitivity in S. aureus compared to E.
coli. Therefore, the use of E. coli as an enumeration tool for
quantifying bacteriophage levels in environmental water samples may
contribute to an understatement of contamination (levels of
bacteriophages present).
As the common use of broad spectrum antibiotics, harsh
chemicals, and irradiation has resulted in the emergence of highly
resistant bacterial strains, new antibacterial treatments are
always necessary. The presence of pili on bacterial cell surfaces
and their demonstrated role in bacterial adherence make them ideal
candidates for vaccines. As the MS2 virus is F pilin selective, an
effective antibacterial treatment based on this selectivity could
be developed. Currently, phage therapy (viruses that specifically
target pathogenic bacteria) for humans is available only at the
Phage Therapy Center in the Republic of Georgia and in Poland.[28]
These institutes have used phage therapy to treat over 1500
patients with bacterial infections, when antibiotic treatment had
failed.[28] This treatment is safe, highly effective, and may be
applied to all patients from whom isolated bacterial strains have
shown sensitivity to specific phages. Phage therapy has been used
to prevent diarrhea caused by E. coli[28] and is of particular
importance to the two pathogens: S. aureus and Pseudomonas
aeruginosa, which have shown sensitivity to specific phages in more
than 80% of the cases.[28]
Studies have already been conducted using the bacteriophage MSa,
testing its activity against S. aureus in mice.[29] Following
simultaneous inoculation with both MSa and lethal or non-lethal
doses of S. aureus, MSa rescued 97% of mice from death and all
non-lethal doses were fully resolved.[29] MSa phage can also
prevent abscess formation and reduce the bacterial load and weight
of abscesses. This suggests a potential use of the phage for the
control of both local and systemic human S. aureus
infections.[29]
These successful treatments will fuel a growing interest in the
use of bacteriophages in medical and commercial practice. This is
already evident. As of January 2, 2007, the United States FDA gave
Omnilytics approval to apply its E. coli O157:H7 killing phage in a
mist, spray or wash on live animals that will be slaughtered for
human consumption.[30]
Although the current experiments yielded interesting
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results with respect to the MS2 bacteriophage and its efficacy
toward S. aureus, the ultimate goal is to develop and successfully
conduct a trial of an anti-viral assay that is cheap, safe, simple,
and allows for high throughput. Studies have shown that A.
barbadensis, C. sinensis, and S. spinescens extracts inhibit viral
growth in human cell lines.[16,31] S. spinescens and C. sinensis
extracts have also shown significant inhibition against the MS2
bacteriophage in our plaque reduction assay. Although inhibition by
A. barbadensis is not as pronounced in the current study, it is
still evident when using this assay. However, it should be noted
that comparing results seen against bacteriophage and viruses in
human cell lines is ambiguous and future investigations using
virally infected human cells lines are needed. By conducting these
studies it will be possible to determine whether or not a direct
comparison between this bacteriophage plaque assay and assays using
viruses in human cell lines exists. If a correlation is seen, using
this MS2 bacteriophage plaque assay as a first step for determining
possible anti viral plants will be a valuable tool.
ACKNOWLEDGMENTS
The MS2 bacteriophage and the F+ and F- Amp+ Escherichia coli
used in these studies were supplied by Dr. Jatinder Sidhu and Dr.
Simon Toze of CSIRO, St Lucia Qld, Australia. The authors thank Dr.
Sidhu and Dr. Toze of CSIRO and Ben Matthews of Griffith University
for the advice and technical assistance given for the development
of this assay for testing plant extracts. Financial support for
this study was provided by the School of Biomolecular and Physical
Sciences, Griffith University.
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Source of Support: School of Biomolecular and Physical Sciences,
Griffith University. Conflict of Interest: None
declared.
Cock and Kalt: Modified MS2 bacteriophage plaque reduction
assay